ISSN 1990-519X, Cell and Tissue Biology, 2008, Vol. 2, No. 6, pp. 614–624. © Pleiades Publishing, Ltd., 2008. Original Russian Text © A.A. Potekhin, I.V. Nekrasova, E. Przybo s´ , M.S. Rautian, 2008, published in Tsitologiya, Vol. 50, No. 6, 2008.
Comparative Description of Macronuclear Electrophoretic Karyotypes of Paramecium primaurelia and Paramecium novaurelia Sibling Species A. A. Potekhina*, I. V. Nekrasovaa, E. Przybo s´ b, and M. S. Rautiana a Faculty
b
of Biology and Soil Science, St. Petersburg State University, St. Petersburg, Russia Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Krakow, Poland e-mail:
[email protected] Received February 11, 2008
Abstract—The macronuclear genomes of two sibling species belonging to the Paramecium aurelia complex, P. primaurelia and P. novaurelia, were analyzed by pulsed-field gel electrophoresis (PFGE). Their electrophoretic karyotypes showed a continuous spectrum of different-sized DNA molecules with a species-specific pattern of banding, which was reproducible and did not change with strain senescence. Thus, P. aurelia macronuclear PFGE profiles could be described by an approach analogous to that used for the description of metaphase chromosome banding patterns. First, well-identifiable regions (size fractions) of the PFGE profile, which could be seen at any resolution, are determined. Then, bands of the second order of resolution (subfractions) can be detected in some regions. The blocks of the first and second order can be utilized as internal markers of the PFGE profile, as they allow for a precise comparison of different PFGE profiles. Such comparative analysis has demonstrated some marked differences in organization of the macronuclear genomes of P. primaurelia and P. novaurelia and a low level of intraspecies polymorphism. It is worth noting that the P. novaurelia strain isolated in the United States was different from all other analyzed P. novaurelia strains of European origin. The nature of banding of the P. aurelia PFGE profiles is discussed. The revealed high order and stability of the macronuclear genome organization makes it possible to search for new approaches to studying the mechanisms of this nontrivial genome formation and maintenance. Further PFGE analysis of the macronuclear genomes of the other Paramecium species in relation with the Paramecium phylogeny may provide insights into directions of evolution of the macronuclear genome in Ciliata. Key words: Paramecium aurelia species complex, sibling species, genome of the macronucleus, karyotype, PFGE. DOI: 10.1134/S1990519X08060084
is eliminated, while the remaining sequences are amplified to different extent. The general scheme of this process is the same for all ciliates, but details of formation (and hence of organization) of the MAC genome differ considerably in various groups of Ciliophora (Katz, 2001; Prescott, 1994).
INTRODUCTION The existence of two differently organized genomes in one cell makes ciliates a unique karyologic model. Micronucleus (MIC) contains dozens of small chromosomes. On the whole, this is the transcriptionally inert nucleus whose main function is transmission of genetic information to daughter cells at the sexual process. Genome of macronucleus (MAC) is actively transcribed determining the cell phenotype. In the course of sexual process (conjugation), MAC is gradually degrading, while MIC undergoes meiosis. The new nuclear apparatus is formed in a series of divisions of zygotic nucleus (synkaryon) formed during the fusion of products of meiotic division of the MIC of partners. During the formation of MAC, the zygotic genome undergoes several essential transformations (see Jahn and Klobutcher, 2002): fragmentation of initial chromosomes occurs; a significant part of genetic material
Pulsed-field gel electrophoresis (PFGE) is a separation of DNA molecules in a gel in the electric field changing by direction. This is currently the only method that allows an analysis of the whole MAC genome. PFGE allows separating DNA molecules measuring from several thousand to twelve million nucleotide pairs (Maule, 1996); this interval includes MAC DNA fragments of Paramecium and Tetrahymena (Altschuler and Yao, 1985; Rautian and Potekhin, 2002). PFGE has been used for study of MAC genomes both of these and of some other ciliates (Lahlafi and Metenier, 1991; Maercker et al., 1999); however, a 614
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detailed analysis and description of PFGE profiles of ciliates have never been performed. PFGE was used for the first time to separate chromosomes of yeast Saccharomyces cerevisiae. Each chromosome of S. cerevisiae migrated in the gel as an individual band (except for two bands, each of which include two chromosomes close in size), which allowed the new term “electrokaryotype” to be proposed (Carle and Olson, 1985). Since for many Protozoa it is difficult or impossible to obtain metaphase plates due to a small size and a great number of chromosomes (Raikov, 1978), the set of PFGE bands can be used as a peculiar equivalent of the traditional, classic karyotype. Since Paramecium MAC contains several hundred amplified DNA fragments with fairly close sizes, their PFGE profile resembles the continuous spectrum of molecules (Caron and Meyer, 1989; Phan et al., 1989; Potekhin et al., 1999). For several species of paramecia, on a continuous background of DNA molecules, more or less distinct bands are identified (Rautian and Potekhin, 2002). The MAC PFGE profile resembles the frequency and distribution of fragmentation sites on MIC chromosomes and different extent of DNA fragments amplification. Previously, we showed that the MAC PFGE profile is a stable characteristic of any strain of Paramecium (Potekhin et al., 1999; Nekrasova et al., 2004). A comparative analysis of the molecular organization of the MAC genome of various morphological species of the genus Paramecium has shown that PFGE profiles of various strains of the same species, even those isolated in regions geographically distant from each other, are very similar, although slight intraspecies differences are sometimes recorded (Rautian and Potekhin, 2002). Thus, the PFGE profile in paramecia is a species-specific characteristic; accordingly, each morphological species is characterized by its own definite pattern of disposition of fragmentation sites on MIC chromosomes. Apart from morphological species, both ciliates in general and representatives of the genus Paramecium in particular are characterized by the presence of the socalled sibling species or genetic species inside morphological species. The sibling species can barely be distinguished at the level of the main morphological characteristics, but are isolated reproductively from each other. The similarity of PFGE profiles of various strains inside morphological species with pronounced differences in electrokaryotypes of various morphological species from one another inevitably raises the question as to how different the PFGE profiles of genetic species (e.g., representatives of the P. aurelia complex of sibling species) are from one another. The MAC genome of one of these species, P. tetraurelia, was recently completely sequenced (Aury et al., 2006). The MAC PFGE profiles of representatives of the P. aurelia comCELL AND TISSUE BIOLOGY
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plex are characterized by the presence of bright bands on the background of the continuous spectrum of DNA molecules (Rautian and Potekhin, 2002). This makes them a convenient model for a comparative study of organization of the MAC genome; the MAC PFGE profile bands can be used as “internal markers.” In this work, we present a detailed description of the MAC PFGE profiles and discuss levels of the intra- and interspecies polymorphism of organization of the MAC genome for two sibling species of the P. aurelia complex, P. primaurelia and P. novaurelia. These species are the most widespread representatives of the P. aurelia complex in Europe (Przybo s´ , 2005). MATERIALS AND METHODS Cultures. To perform this work, 14 strains of P. primaurelia and P. novaurelia were used. P. primaurelia: 156 (New Haven, CT, United States); 60 (Burlington, VT, United States); AZ9-3 (Astrakhan Reserve, Russia); AZ11-13 (Astrakhan Reserve, Russia); AZ12-19 (Astrakhan Reserve, Russia); AZ15-8 (Astrakhan Reserve, Russia); V7-6 (Volgograd region, Russia); KK2-7 (Kaliningrad region, Russia). Strains from the United States were provided by Prof. H. Schmidt (Kaiserslauten University, Germany). P. novaurelia: 91 TR-9 (St. Petersburg, Russia); 90 VL4-8 (Vladimir, Russia); Iz-16 (Izmail, Ukraine); Nov (Germany; provided by Prof. H.-D. Görtz, Stuttgart University, Germany); V9-1 (Volgograd region, Russia); AB8-22 (Boston, MA, United States). All these strains were maintained in the Collection of Paramecium Strains at the Laboratory of Protozoan Karyology of the Faculty of Biology and Soil Science, St. Petersburg State University. The species specificity of the used strains was determined by crossing with tester strains (strain 90 of P. primaurelia and strain 510 of P. novaurelia). Cultivation of the Paramecium strains was performed by the standard method (Sonneborn, 1970). The nutrition medium was a lettuce broth inoculated with bacteria Klebsiella cloacae. Synchronization of paramecian cultures. Synchronous cultures of paramecia were obtained by the method described in the literature (Sonneborn, 1970). With the aid of a microscope, cells that completed autogamy (those having two developing MAC) were isolated into individual microaquaria. The vegetative offspring of each of these cells was fed from 800 µl of nutrition medium per cell; i.e., the culture was maintained at excessive feeding. Under such conditions, P. aurelia divide at a constant high rate (two to three fissions/day) and autogamy in the cells begins simultaneously after 23–27 fissions (cells of the culture maintained under standard conditions without excessive
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feeding normally begin to enter autogamy separately after 18–20 fissions). One cell of the culture was isolated per day and transferred into a new microaquarium, which allowed for the fairly precise recording of the frequency of divisions of all cells of the culture. Every two days, several hundred cells were stained by standard Feulgen’s method (Pearse, 1960), which allowed us to determine precisely whether the culture entered the sexual process, as well as to reveal the degree of its synchronism. To make preparations for pulsed-field gel electrophoresis (PFGE), 10–20 ml of paramecian cultures were used in which the number of cells achieved 103– 104 per 1 ml. Through mild centrifugation, the cells were concentrated in the volume of 0.3–0.6 ml. Block insertions for PFGE (a mixture of cell suspension and agarose) were prepared by the method described previously by us (Potekhin et al., 1999). As a size marker for PFGE, chromosomes of the yeast Saccharomyces cerevisiae of the strain 15V-P4 were used; their size was previously determined at our laboratory (Timofeeva and Rautian, 1997). Preparations of yeast DNA were prepared by the standard method (Carle and Olson, 1985). To perform PFGE, we used a self-made apparatus (Mezhevaya et al., 1990) that results in the creation and alternation of two electrical fields with a reorientation angle of 120°. PFGE was performed in 1% agarose gel (Sea Kem, FMC, United States) prepared in 0.5× TBE buffer. The thickness of the gel amounted to 4 mm. Electrophoresis was performed in 0.5× TBE buffer at a temperature of 16°C. The voltage of the field was 10 V/cm. Under these conditions, the optimal DNA separation of MAC of P. aurelia was achieved in the following PFGE regime: 30 s for 5 h, 60 s for 7 h, 90 s for 10 h, 120 s for 13 h, 150 s for 8 h (the so-called “pulse time,” i.e., the time of the action of each electrical field in seconds; the duration of the operation of the apparatus at this pulse time in hours). Southern hybridization. The PFGE-separated DNA was transferred to nylon membranes (Amersham Pharmacia Biotech Nylone Membranes, United Kingdoms) by the capillary method according to the protocol of the membrane manufacturer and was immobilized by ultraviolet radiation. The probe for hybridization was obtained in the polymerase chain reaction (PCR) by adding DIG-11-dUTP (Roche Diagnostics, Mannheim, Germany) into the mixture for PCR. The sequence from the macronuclear repeat P126 (Forney and Rodkey, 1992) was used as the probe for hybridization, while DNA of P. primaurelia and P. novaurelia that had been totally isolated by the standard method of phenol-chloroform extraction served as a template for PCR (20–50 ng per reaction). Oligonucleotides 5'-tctatccgtttatgggatgt-3' and 5'-cactaccggatgctaatgta-3'
(Syntol, Russia) chosen from the sequence of the repeat P126 P. tetraurelia (GenBank M96642) were used as primers. The PCR parameters for the probe synthesis are as follows (Techne thermocycler, United Kingdom): 94°C for 45 s, 55°C for 45 s, 72°C for 45 s; 30 cycles with the melting time increased to 4 min in the first cycle and the annealing time increased to 3 min in the first three cycles. Yield of PCR was controlled by electrophoresis in 1% agarose gel in 0.5× TBE buffer. Southern hybridization was performed by the standard procedure of the membrane manufacturer at 61°C. Signal was detected by using a kit for colorimetric detection of digoxygenin (Roche Diagnostics, Mannheim, Germany), following manufacturer’s recommendations. Thermal washings of membranes after hybridization were performed in 0.5× SSC at a temperature 1°C higher than the temperature of hybridization. RESULTS Description of PFGE profiles of P. primaurelia and P. novaurelia. By the PFGE, we analyzed 14 strains of two species of the P. aurelia complex, P. primaurelia and P. novaurelia. The size of DNA molecules of MAC of these ciliates varies from approximately 75 to 1100 kb. Moreover, it is sometimes possible to reveal the presence of high-molecular DNA fragments (around 2500 kb), which form an accessory, individual, poorly pronounced band (for instance, see Fig. 1; shown by arrow). The PFGE profiles of all analyzed strains, like all other representatives of the P. aurelia complex (Nekrasova et al., 2004), are characterized by the presence of bands on the background of the continuous spectrum of DNA molecules (Fig. 1). These bands can be used as reliable “internal markers” of the PFGE profiles of P. aurelia. This banding of the P. aurelia PFGE profiles gave us the idea to use the hierarchical nomenclature of blocks in their description by analogy with the system used in the description of G-banding of mammalian metaphase chromosomes (Sumner, 1990). First, we identified the most pronounced blocks (DNA size fractions) of the studied PFGE profiles, i.e., those that are clearly seen at any parameters of performance of the experiment. For the PFGE profiles of all studied strains, nine of these blocks can be identified, which we refer to as blocks of the first order. Some blocks are complex, consisting of two or more bands which, in some PFGE runs, can be separated clearly or defined within a background of DNA. These bands are referred to as the bands of second-order resolution. Since, as was said above, the PFGE profiles of Paramecium represent a spectrum of DNA fragments of different sizes, the description of electrokaryotypes based only on dimensional characteristics can lead to a great number of inaccuracies, especially when comparing results obtained by different authors in various experiments. CELL AND TISSUE BIOLOGY
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P
II
P. novaurelia
I
1 2 3 4.1 4.2 5.1 5.2 5.3 6.1 6.2 7.1 7.2 7.3 7.4
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P
Sc
N
I
II
1 2 3
1 2 3
4 5 6
4 5 6
7
7
N Sc
1 2 3 4.1 4.2 5.1 5.2 6.1 6.2 7.1 7.2 7.3 7.4 7.5 7.6
7.5
8 9
8 9
8 9
8 9
Fig. 1. PFGE profiles of P. primaurelia (P) and P. novaurelia (N) and their representation in the form of blocks of the first (I) and of the second (II) orders. Sc are chromosomes of yeast S. cerevisiae. Arrow indicates the high-molecular weight band (about 2500 kb).
The formalized description of electrokaryotypes, which we have proposed, facilitates comparative analysis of the obtained data. We developed a detailed hierarchical description of electrokaryotypes for two species of the P. aurelia complex, P. novaurelia and P. primaurelia. P. primaurelia. Eight strains of P. primaurelia were studied. PFGE profiles of all analyzed strains represent the continuous spectrum of DNA molecules of different sizes; among them, nine bands and zones, i.e., blocks of the first orders (see table and Fig. 1), can be revealed. The three upper blocks (blocks 1, 2, and 3) are never separated into blocks of the second order; block 2 is always brighter than blocks 1 and 3. Blocks 4 and 6 can be divided into two blocks of the second order each. Block 5 on PFGE profiles of some strains (for instance, of strain 156) is divided into two blocks of the second order (Fig. 2, lane 5), whereas on PFGE profiles of other strains (AZ15-8 and AZ11-13) inside the block 5, three blocks of the second order can be identified (Fig. 2, lanes 1, 2). Inside block 7, i.e., an expanded zone of the PFGE profile formed by DNA molecules measuring approximately 500–100 kb, five blocks of the second order always can be revealed. In all studied strains, two distinct lower blocks, 8 and 9, are also clearly seen that are not separated into blocks of the second order. On the whole, PFGE profiles of all strains of P. primaurelia are very similar, though some intraspecies differences do exist. Thus, in strain 60, blocks 5 and 6 are clearly separated into two blocks of the second order each; they are so distinct that they can even be considered blocks of the first order (Fig. 2, lane 4; denoted with arrows); CELL AND TISSUE BIOLOGY
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this distinguishes strain 60 from all remaining strains of P. primaurelia. P. novaurelia. Six strains of P. novaurelia were studied. The PFGE profile of P. novaurelia is the clearest as compared to PFGE profiles of other species of P. aurelia (Nekrasova et al., 2004); it also contains nine blocks of the first order (see table and Fig. 1). Three P. primaurelia 1 2 3 4
5
6
P. novaurelia 7 8 9 10 kb 2000 1120 800 590 430 250
Fig. 2. PFGE profiles of five strains of P. primaurelia (lanes 1–5) and of four strains of P. novaurelia (lanes 7–10). Lanes: 1, AZ15-8; 2, AZ11-13; 3, V7-6; 4, 60; 5, 156; 6, chromosomes of S. cerevisiae; 7, 91TR-9; 8, Iz-16; 9, V9-1; 10, 90 VL4-8. Arrows indicate bands of the PFGE profile, which distinguish strain 60 from other strains of P. primaurelia.
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Approximate size of DNA molecules forming blocks of the first and second orders in electrokaryotypes of P. novaurelia and P. primaurelia. Blocks are exposed in the table in correspondence with sizes of their forming DNA molecules, so it is easy to compare characteristics of the two species P. novaurelia
P. primaurelia
Blocks of electrokaryotype of the first order
Approximate size of DNA molecules, of the second order kb
Blocks of electrokaryotype of the first order
Approximate size of DNA molecules, of the second order kb
1
–
1060–1100
1
–
1010–1030
2
–
1000–1020
2
–
950–980
3
–
900–930
3
–
890–930
4
4.1
820
4
4.1
820–840
4.2
780–800
4.2
770–800
5.1
700–740
5
6 7
5 5.1
690–700
5.2
660–690
5.2
640–650
5.3
630–650
6.1
590–630
6.1
590–630
6.2
570–590
6.2
570–590
7.1
520
7.1
510–550
7.2
500
7.3
470
7.2
470
7.4
430
7.3
410
7.5
380
7.6
330
7.4
340
7.5
250
6 7
8
–
105
8
–
90
9
–
75
9
–
65
upper bands, blocks 1, 2, and 3, are very bright and are not divided into blocks of the second order; block 3 is usually the brightest. Then, there are three less compact blocks (4, 5, and 6), each of which can be divided into two blocks of the second order. In block 7, no less than five blocks of the second order can be identified. These blocks are followed by two distinct bands that we designate blocks 8 and 9. This pattern of the PFGE profile is characteristic of all five analyzed European strains (Fig. 2, lanes 7–10). However, the PFGE profile of the only existing strain of P. novaurelia of American origin AB8-22 (Przybo s´ et al., 2007) differed from the rest (Fig. 3). Thus, block 5 in the PFGE profile of this strain is separated into three distinct blocks of the second order, whereas all European strains of P. novaurelia are characterized by the presence of two blocks of the second order inside the block 5. Furthermore, of the first three blocks, the brightest of all of the European strains turns out to be block 3, while, in the case of block AB8-
22, block 2 is the brightest. It is worth noting that, in the case of P. primaurelia, the revealed differences of PFGE profiles did not correlate to the geographic origin of the strains. In spite of the revealed intraspecies differences, we can claim that, for both studied species of the P. aurelia complex, PFGE profiles of all strains inside the species are very similar to one another (except for strain AB822) and differ essentially from PFGE profiles of representatives of the other species (Fig. 2); i.e., based on the figure of the PFGE profile, we can unequivocally distinguish the representatives of P. primaurelia from representatives of P. novaurelia. In all P. novaurelia (again, except for strain AB8-22), the brightest out of blocks 1, 2, and 3 is block 3, while in all representatives of P. primaurelia, without exception, the brightest is block 2. Blocks 4, 5, and 6 in P. novaurelia are very clear and unique, whereas, in representatives of P. primaurelia, they are more blurred. CELL AND TISSUE BIOLOGY
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9a
9e
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2
3
4
5
6
7
619
8 kb
kb
2000
>2000 1120 1120
800 590 430
800
250
590
430 250
Fig. 3. PFGE-profiles of the American (AB8-22; lane 9a) and of one of the European (Iz-16; lane 9e) strains of P. novaurelia. Arrows show blocks differing in the strains. Sc are chromosomes of yeast S. cerevisiae.
Effect of senescence of MAC on PFGE profiles. Autogamy, the sexual process occurring inside one cell in the absence of partner, is a characteristic feature of all species of Paramecium aurelia complex (Wichtermann, 1986). In the norm, cells of a strain undergo autogamy after approximately every 25–30 fissions. If the culture is not synchronized, all cells of the strain at each time moment differ in the number of their divisions after the last autogamy. During each autogamy, the old MAC is destroyed. In autogamonts, MAC is formed de novo from products of mitotic division of synkaryon. Accordingly, various cells of one strain differ in the age of their MAC. Since MAC divides amitotically and different amounts of DNA remain in daughter cells (Berger, 2001), with MAC senescence, changes can theoretically appear in the molecular composition of its genome. This means that, for each P. aurelia strain, the cell line obtained from one isolated cell is a clone related to the MIC genome but a population related to the MAC genome. As a result, in the case of the use of unsynchronized cultures in experCELL AND TISSUE BIOLOGY
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Fig. 4. PFGE profiles of the synchronized P. primaurelia strain AZ9-3 at different stages of MAC senescence. Lanes: 1, 15 fissions after autogamy, 2, 20 fissions, 3, 27 fissions, 4, 70% cells entered autogamy (several products of MIC division are observed within them), 5, formation of new MACs starts in autogamonts, 6, large anlages of new MAC and small fragments of old MAC in postautogamous cells, 7, 10 fissions after autogamy, 8, chromosomes of S. cerevisiae; arrow on pages denote high-molecular weight DNA in PFGE profiles.
iments, various cells of the strain will differ in the molecular composition of their MAC genome. Thus, it needed to be checked whether the MAC senescence produces detectable PFGE profile changes capable of yielding biased results. We synchronized the P. primaurelia strain AZ9-3 and compared PFGE profiles of cultures composed of cells with young MACs (immediately after autogamy), mature MACs (in the middle of the life cycle, 10–20 fissions after autogamy), old MACs (27 fissions, directly before autogamy), and the cells that perform autogamy. It was shown that PFGE profiles of cells after 10, 15, 20, and 27 fissions after autogamy did not differ from each other (Fig. 4). The only difference were that, in PFGE profiles of early postautogamy and cells after 15 divisions after autogamy, a high-molecular band (Fig. 4, denoted by arrow on margin) was revealed that, with further cell divisions, became progressively less pronounced. In some of our used preparations of nonsynchronized strains, a similar band was also clearly observed.
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2
(b) 3
4
5
1
2
3
4
5
kb >2000 1120
800 590 430 250
Fig. 5. Results of PFGE separation (a) and Southern hybridization with the probe P126 (b) of total DNA of two strains of P. primaurelia (lane 1, strain 60, 2, strain AZ11-13) and two strains of P. novaurelia (lane 3, strain 91TR-9, 4, strain Iz-16). Lane 5, chromosomes of S. cerevisiae. Arrows denote blocks of PFGE profiles, which do not produce hybridization signal.
Analysis of PFGE profiles by Southern hybridization. As an additional criterion in the comparative analysis of PFGE profiles of P. primaurelia and P. novaurelia strains, we performed Southern hybridization of DNA separated by PFGE of different strains of two studied species with the sequence of the P126 repeat. This repeat was revealed in subtelomeric regions of most DNA molecules of MAC of P. tetraurelia (Forney and Rodkey, 1992). Thus, the use of the P126 repeat allows us to obtain a pattern of hybridization with numerous signals, which can be considered another characteristic of the polymorphism of genomes. We have obtained a complex pattern of hybridization for both studied species; at least 12 individual signals in the case of P. novaurelia and 10 signals in the case of P. primaurelia (Fig. 5). For both species, the background hybridization also was observed. On the whole, the obtained hybridization pattern greatly resembled the picture of the PFGE profile itself, which confirms the initial data regarding the localization of the P126 repeat on many different DNA fragments of MAC (Forney and Rodkey, 1992). Nevertheless, not all MAC DNA contain the P126 repeat: for instance, the DNA molecules forming bright blocks 1 and 6.2 in PFGE profiles of P. novaurelia do not produce the hybridization signal with the P126 sequence (Fig. 5; denoted by arrows). The obtained hybridization
pattern, like the MAC electrokaryotype itself, is species specific. No intraspecies differences between various strains have been detected, but all representatives of P. primaurelia obviously do differ in their hybridization pattern from the representatives of P. novaurelia. DISCUSSION Since the amount of DNA in MIC in P. aurelia accounts for less than one percent of the total amount of DNA in the cell (Raikov, 1978), nearly all of the DNA separated with aid of PFGE is DNA from the MAC. The MAC genome of another Paramecium species related to P. aurelia, P. caudatum, contains about 300 DNA fragments of different sizes (Potekhin et al., 1999), amplified to different extents. As a result, it becomes impossible to separate individual DNA molecules of MAC in PFGE as discrete bands. Indeed, the PFGE profile of all representatives of P. aurelia represents a continuous spectrum of molecules; nevertheless, its background contains distinct individual zones and bands. A similar picture was also observed by the authors of the first works on separation of DNA of Paramecium aurelia by the PFGE (Caron and Meyer, 1989; Phan et al., 1989); however, we managed to obtain the PFGE profiles with better resolution. We were able to reveal some blocks only under certain regimes of the PFGE performance, which handicaps CELL AND TISSUE BIOLOGY
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the direct description and comparison of the PFGE profiles obtained in different experiments. This problem can be resolved by our proposed principle of the hierarchical description of PFGE profiles; the pattern of the disposition of the first-order blocks in the PFGE profile is constant characteristics of a strain and, most likely, of the genetic species inside the P. aurelia complex independent of specific peculiarities of experiment performance. The only band that cannot always be revealed in PFGE profiles is the band formed by the high-molecular DNA (>2000 kb). Our experiments performed on synchronized cultures to study the effect of the senescence of MAC cells on the PFGE profiles have shown that this band is highly pronounced on PFGE profiles of postautogamy cells; however, upon passing through further divisions by the cells, it gradually disappears, finally becoming totally absent. It is quite probable that, even in the synchronized culture, autogamy expands in time and a high-molecular band is formed by cells in which, at this particular moment, DNA processing is taking place. The percentage of these cells in synchronized culture decreases and the band becomes increasingly less pronounced. Another probable explanation for the appearance of this high-molecular band in PFGE profiles is the long presence of underprocessed DNA in maturing MACs. The DNA processing is still likely to continue in cells that, after passing through the sexual process, begin vegetative division, although this contradicts the commonly accepted opinion that the formation of the genome is completely finished at the moment when the MAC acquires its ability to divide (Prescott, 1994). With cell fissions, the amount of such molecules in each cell will decrease and gradually will become too insignificant to be detected by the PFGE. Besides, this band can contain MIC chromosomes whose sizes are too large for separation by the PFGE. However, as noted above, the relative DNA content in MIC is very low, perhaps even too low for stable detection in PFGE profiles. We also cannot rule out the possibility that the composition of the highmolecular band may also include DNA of alimentary bacteria despite the fact that the DNA samples were prepared from the paramecia cleansed of the nutrition medium, so their digestive vacuoles were nearly empty. Anyway, this high-molecular band cannot be considered a constant characteristic of P. aurelia electrokaryotypes; therefore, we do not include it in the blocks identified during the hierarchical description of PFGE profiles. It is important to note once more that, except for this high-molecular band, we have not revealed any changes in the PFGE profiles connected with the senescence of cellular MACs. The unique process of MAC division, amitosis, leads to a high probability of random changes in the number of copies of various DNA molecules (Berger, 2001). Theoretically, this might lead to CELL AND TISSUE BIOLOGY
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global changes in the MAC genome molecular composition and, accordingly, in the PFGE profile. Nevertheless, results of our experiments with use of synchronized cultures indicate unanimously that such changes do not take place. Previously, it was shown (Gilley and Blackburn, 1994) that, during the aging of a synchronized culture of P. tetraurelia in which autogamy was inhibited artificially, a gradual degradation of DNA molecules occurred; the hybridization signal, which, in postautogamy cells was seen as a distinct band, became blurred after cells with 50 or more fissions had been washed out. In addition to the initial band, an additional smear appeared. This means that a part of the DNA molecules carrying a target for hybridization became shorter due to their gradual degradation. The described changes might have been detected in a PFGE profile by leading to the appearance of an additional background of degrading DNA molecules of different sizes and, accordingly, to a decrease in the clearness of bands and blocks. However, it is only possible to observe this process, i.e., to modulate real aging, by preventing the ciliates from entering into autogamy and renovating the MAC genome throughout several dozen cycles of vegetative divisions. In normal laboratory cultures of P. aurelia in the absence of agents inhibiting the sexual process, the number of divisions that the postautogamy cell passes through until the next autogamy does not exceed 25–30, even under conditions of excess of food (Sonneborn, 1970). Under these conditions, the marked degradation of the MAC DNA molecules does not yet occur (Gilley and Blackburn, 1994) and the PFGE profile remains unchanged. Thus, to obtain objective data by PFGE, it is not necessary to use synchronized cultures, as, under standard conditions, the possible changes in the MAC genome during the senescence of this nucleus do not produce a principal effect on its molecular composition and, hence, on the PFGE profile. The origin of bands in the P. aurelia PFGE profiles can be connected with both the fact that different MAC DNA fragments close by their sizes are grouped at separation in a gel and the hyperamplification of some DNA fragments in MAC. Thus, the upper band in the P. tetraurelia PFGE profile has been shown to be formed by a single, many-copy, 984.6-kb DNA molecule (Zagulsky et al., 2004). Since the block 1 of the P. primaurelia and P. novaurelia electrokaryotypes is expressed equally brightly and located in approximately the same size area, it can be suggested that this block is also formed by a single hyperamplified DNA fragment. In 2006, the complete sequencing of the P. tetraurelia MAC genome was completed (Aury et al., 2006; http://paramecium.cgm.cnrsgif.fr/db/index). The entire MAC genome was presented as a set of scaffolds assembled from overlapping sequenced DNA sequences terminated by telomeres at both ends. Thus,
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each scaffold corresponds to an individual MAC DNA fragment. By comparing the sizes indicated for each scaffold and the approximate sizes of the DNA molecules forming some particular electrokaryotype bands, it is possible to explain the probable origin of at least some bands within PFGE profiles of the P. aurelia representatives. Thus, the first three scaffolds of the P. tetraurelia MAC genome have sizes of 984, 950, and 893 kb, respectively. The approximate size of the DNA molecules forming the first three blocks of the P. primaurelia electrokaryotype (see table) is, respectively, 1020, 960, and 910 kb, which reveals the following correspondence: block 1: P. primaurelia, scaffold 1 P. tetraurelia; block 2: scaffold 2; block 3: scaffold 3. In the case of P. novaurelia, the approximate sizes of the DNA molecules that form the first three electrokaryotype bands (see table) somewhat exceed the sizes of the first three P. tetraurelia “scaffold” (block 1, 1080 kb; block 2, 1010 kb; block 3, 915 kb); however, it is probable that, in this case, each of these three blocks is also formed by the same type of DNA fragments. It is difficult to reveal further correspondence without additional analysis, as the differences in scaffold sizes are too insignificant; differences are also possible in the organization of the MAC genome of representatives of different species. This origin of the first three blocks of PFGE profiles of P. primaurelia and P. novaurelia is also confirmed by the results of hybridization with the sequence P126. In block 1 of the P. novaurelia PFGE profile, the hybridization signal is absent. Since the subtelomeric repeat P126 is present in the majority of MAC DNA molecules, the probability of grouping several types of DNA molecules, none of them containing this repeat, is not very high. Block 1 of the P. novaurelia electrokaryotype is no less intensive than blocks 2 and 3, which emit a bright signal at hybridization. Thus, the obtained data confirm the statement that we claimed earlier, i.e., the obtained PFGE profiles reflect the native spectrum of the DNA molecules represented in the MAC genome of paramecia (Potekhin et al., 1999; Rautian and Potekhin, 2002) and do not depend on the age or physiological state of the culture. The revealed orderliness and stability of the organization of the MAC genome, an unusual nucleus that, until recently, due to the absence of its genome analysis methods, was called a “sack with genes” (Prescott, 1994), has given us a new perspective that will allow us to approach the analysis of how this untrivial genome is formed and maintained from new angles.
Even phylogenetically close morphological species of Paramecium differ astonishingly in the molecular organizations of their MAC genome (Rautian and Potekhin, 2002). These differences are too large for there to be any possibility of tracing any tendencies of changes in the program of formation of the MAC genome in evolution of Paramecium. At the same time, PFGE profiles of various syngens in some species (syngens on the whole can be considered as an early stage of divergence of species) are characterized by a high similarity (Potekhin et al., 1999; Rautian and Potekhin, 2002). The complex of P. aurelia species is an excellent model for performing comparative studies, as it includes 15 syngens that eventually transformed into true sibling species (Sonneborn, 1975; Aufderheide et al., 1983). In the present work we have shown that MAC genomes of P. primaurelia and P. novaurelia are formed in a similar pattern, i.e., the most pronounced blocks of the first order in the analyzed strains coincide. On the other hand, some differences were revealed, not only between representatives of the P. primaurelia and P. novaurelia species, but even between various strains inside each species. In the case of P. primaurelia, we did not reveal regularities in the appearance of intraspecies polymorphism, while, in the case of P. novaurelia, the intraspecies differences turned out to be characteristic of the strains isolated from the geographically reliably uncoupled populations. Nevertheless, these data are not sufficient to judge the causes of the appearance of intraspecies polymorphism from organization of MAC genome and about the potential role of geographical isolation in this process. In recent years, several molecular-phylogenetic dendrograms involving the entire Paramecium genus or several of its morphospecies (Strueder-Kypke et al., 2000; Hori et al., 2006; Hoshina et al., 2006) were published. Further comparison of MAC genomes of various paramecian species and the data of these analyses with the phylogeny of the genus can shed light on directions of evolution of the formation and organization of the MAC genome in Ciliates. ACKNOWLEDGMENTS The authors are grateful to Prof. H.-D. Görtz and Prof. H. Schmidt for the provided Paramecium strains. The work is supported by the Russian Foundation for Basic Research (project 06-04-49504), Russian Ministry of Education and Science (grant RNP 2.2.3.1.4148, grant A04-2.12-655), grants of St. Petersburg Government (to A.P. and I. N.),. The work was performed with use of equipment of the CHROMAS Center. CELL AND TISSUE BIOLOGY
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COMPARATIVE DESCRIPTION OF MACRONUCLEAR ELECTROPHORETIC KARYOTYPES
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